This invention relates generally to the field of growth factors and specifically to Connective Tissue Growth Factor (CTGF) and methods of use thereof.
A. The Role Of Growth Factors in Bone and Cartilage Formation
Bone And Cartilage Formation. The formation of tissue and organs in all multicellular organisms that arise from a single fertilized egg requires the differentiation of specialized cell types from non-differentiated stem cells. As embryogenesis proceeds, more highly specialized cell types and complex structures are formed. Currently, however, little concrete information is available on the identification of the specific factors or the mechanism of action of these factors on skeletal or cartilage formation in vertebrate animals, including humans.
There are two common types of bone formation in the mammalian system: intramembranous ossification and ehdochondral ossification. The formation of the bones of the skull are an example of intramembranous ossification. There, mesenchymal cells from the neural crest interact with the extracellular matrix of the cranial epithelial cells and form bone. Hall, Amer. Sci. , 1988, 76174-181. Mesenchymal cells condense into small islands and differentiate into osteoblasts and capillaries. The osteoblasts secrete a specific type of extracellular matrix, (osteoid) which is capable of binding calcium salts.
Endochondral ossification is the process by which the long bones of the axial skeleton (arms and legs), and the vertebra and ribs form. Hall, supra. During this process the formation of bone occurs via a cartilaginous tissue intermediate stage. In mammals, the long bones form from certain mesenchymal cells in the embryonic limb buds. These cells form chondrocytes, and secrete a cartilaginous matrix. Other surrounding mesenchymal cells form the perichondrium (ultimately, the periosteum). In some cases, chondrocytes adjacent to the region where chondrocytes are proliferating and forming differentiate into hypertrophic chondrocytes.
Hypertrophic chondrocytes produce a different type of matrix, and alter their tissue orientation to form the physis. The structure of the physis is arranged in multiple 30 cellular columns composed of zones of cellular hypertrophy, proliferation, ossification and vascularization. Hall, supra; Gilbert, xe2x80x9cTranscriptional regulation of gene expression,xe2x80x9d DEVELOPMENTAL BIOLOGY, 5th ed. Sinaur Assoc., p. 387-390 (1994). This results in a gradation of cell transformation from chondrocytes to osteoblasts which form the mineralized-bone.
Endochondral ossification is an active, ongoing process that occurs in mammals during the growth from infant to adult. The differentiation of mesenchymal cells to chondrocytes, their proliferation and replacement by osteoblasts are dependent on growth factors (including the TGF-xcex2 family), and on the mineralization of the matrix. Tuan, 1984, J. Exp. Zool. (suppl.) 1:1-13 (1984); Syfestad and Caplan, 1984, Devel. Biol. 104:348-386.
With regard to connective tissue, it is felt that all skeletal elements in mammals are derived from a single stem cell that is capable of differentiating into the specific cell types that compose muscle, cartilage, bone and tendon. These cells also appear to be capable of differentiating into adipose tissue.
The Relevant Art Related To Growth Factors And The Formation Of Bone And Cartilage. Prior to the present invention, it was known generally that growth factors comprise a class of secreted polypeptides that stimulate target cells to proliferate, differentiate and organize developing tissues. Typically, a growth factor""s activity is dependent on its ability to bind to specific receptors, thereby stimulating a signaling event within the cell. Examples of some well-studied growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor beta family (TGF-xcex2), transforming growth factor alpha (TGF-xcex1), epidermal growth factor (EGF), and fibroblast growth factors (FGF).
Effect Of TGF-xcex2 On Chondrocyte Growth, Differentiation and Cartilage Formation. The TGF-xcex2s play a role in chondrogenesis. As previously reported, TGF-xcex21 and TGF-xcex22 increase chondrogenesis in embryonic rat mesenchymal cells (Seyedin,et al.., 1987, J. Biol. Chem. 262: 1946-1947), and either isoform can induce formation of chondroblasts from murine muscle mesenchymal cells in culture. Seyedin. et al., 1986, J. Biol. Chem. 261: 5693-5695. Application of the TGF-xcex2s to murine embryonic prechondroid tissues increases differentiation of mesenchymal cells, production of proteoglycans, and replication of chondroblasts. Centrella, et al., 1994, Endocrine Reviews 15:27-38; Thorp and Jakowlew, 1994, Bone 15: 59-64.
Using in-situ hybridization, decreased levels of TGF-xcex23 were found in the growth plates of animals with three separate disorders where chondrocytes cease to differentiate. Id. In organ cultures of bovine articular cartilage, type II collagen and proteoglycan synthesis were increased after TGF-xcex2 administration. Morales and Roberts, 1988, J. Biol. Chem. 263: 12828-12831. In contrast, the TGF-xcex2s have been shown to decrease expression of type II and type X cartilage-specific collagens, synthesis of chondrocyte proteoglycans, and activity of alkaline phosphatase in cultured chondroid cells. Mundy. xe2x80x9cThe effects of TGF-xcex2 on bone,xe2x80x9d Clinical Applications of TGF-xcex2., 1991, Wiley Chichester, Ciba Foundation Symposium 157: 137-151. Rabbit growth plate chondrocyte differentiation is inhibited by TGF-xcex2, while growth plate chondrocyte mitogenesis is increased. Kato, et al., 1988, Proc. Natl. Acad. Sci. USA 85: 9552-9556. In addition, large concentrations of TGF-xcex21 or TGF-xcex22 added to an osteoinductive model favor cartilage, rather than the preference for bone formation, when smaller doses are used. Mundy, supra. This accumulation of apparently contradictory data has hindered efforts to define a function for the TGF-xcex2s in chondrogenesis.
The Bone Morphogenic Proteins And Bone Formation. A family of proteins termed the bone morphogenetic proteins (BMP""s are capable of inducing ectopic bone formation in certain mammalian species. With the exception of BMP-1, which encodes a metalloprotease, all of these proteins have structures that are related to TGF-xcex2. However, it is not known which, if any of the BMP""s are responsible for the regulation of bone formation during normal embryogenesis.
BMP""s were first isolated from demineralized bone as factors that induced bone at extra skeletal ectopic sites. Three peptides were originally identified as BMP-1, BMP-2A, and BMP-3. Celeste, et al., 1990, Proc. Natl Acad. Sci. USA 87: 984.3-9847; Kubler and Urist, 1990, Clin. Orthopedics and Rel. Res. 258: 279-294. The latter two BMPs are members of the TGF-xcex2 superfamily. Subsequently, five more closely related members of the BMP group have been identified and cloned. BMP-5, BMP-6, and BMP-7 are most similar to vgr/60A, while BMP-2 and BMP-4 are more similar to Decapentaplegic. Both vga/60A and Decapentaplegic are Drosophila genes that control dorsal/ventral axis pattern formation. Hoffman, 1992, Mol. Repro and Dev. 32: 173-178.
In-situ hybridization has localized BMP""s gene transcription to areas of bone formation in the limb bud at specific times during development, suggesting a physiologic role. The BMPs induce adventitial post-fetal mesenchymal cells to switch from fibrogenetic to chondroosteoprogenetic patterning. Kubler and Urist, supra. Several lines of data suggest the BMPs may act synergistically with TGF-xcex2s to initiate the cascade of osteoinduction in-vivo. In murine subcutis, TGF-xcex21 enhances the production of ectopic bone by most BMPs. BMP-6 (also known as VGR-1) is expressed in hypertrophic cartilage at the same time and in the same areas as the TGF-xcex2s, and is associated with collagen type X expression. See, Celeste, et al., supra.
The addition of TGFxcex2-2 to bone explants which have been treated with either BMP-2 or BMP-3 results in increased osteoinductive activity and an increased ratio of cartilage to bone when compared to either factor alone. Bentz, et al., 1991, Matrix 11:269-275. However, the synergistic effect of these proteins by the TGF-xcex2s is not universal. TGF-xcex21 has been shown to directly decrease BMP-2 expression in fetal rat calvaria cultures. Harris, et al., 1994, J. Bone and Mineral Res. 9: 855-863. Since BMP-2 is apparently important in bone cell differentiation, it has been suggested that TGF-xcex21 may be acting as a switch to monitor the differentiation fates of chondro- or osteo-blastic precursors.
Other Factors Found To Be Expressed In Developing Tissue. Cyr61 is a growth regulator which has been found to be expressed in developing mouse embryo and extraembryonic tissues. O""Brien and Lau, 1992, Cell Growth Differ. 3:645-654. Cyr61 is related to but distinct from CTGF and prior to the instant invention, the specific activity of Cyr61 was not known.
B. The Role of Growth Factors in Wound Healing Platelet Derived Growth Factor and Wound Healing.
PDGF is a dimeric molecule consisting of an A chain and a B chain. The chains form heterodimers of homodimers and all combinations isolated to date are biologically active. With respect to the factor""s activity, PDGF has been characterized as a cationic, heat-stable protein found in the xcex1-granules of circulating platelets. The molecule has been further characterized as a mitogen and a chemotactic agent for connective tissue cells such as fibroblasts and smooth muscle cells.
Because of PDGF""s biological activity and release during wound healing, PDGF has been identified as a growth factor involved in wound healing, as well as pathological conditions showing on overproduction of connective tissue, including atherosclerosis and fibrotic diseases.
It has been hypothesized that growth factors other than PDGF may play a role in the normal development, growth, and repair of human tissue.
TGF-xcex2 And Wound Healing. The formation of new and regenerating tissue requires the coordinate regulation of various genes that produce both regulatory and structural molecules which participate in the process of cell growth and tissue organization. As with bone induction, it appears that TGF-xcex2 plays a central regulatory component of this process. TGF-xcex2 is released by platelets, macrophages and neutrophils which are present in the initial phases of the repair process. TGF-xcex2 can act as a growth stimulatory factor for mesenchymal cells and as a growth inhibitory factor for endothelial and epithelial cells. It has been suggested that the growth-stimulatory action of TGF-xcex2 appears to be mediated via an indirect mechanism involving the induction of other genes including growth factors such as PDGF.
Several members of the TGF-xcex2 superfamily possess activities suggesting possible applications for the treatment of cell proliferative disorders, such as cancer. In particular, TGF-xcex2 has been shown to be potent growth inhibitor for a variety of cell types (Massague, 1987, Cell 49:437), MIS has been shown to inhibit the growth of human endometrial carcinoma tumors in nude mice (Donahoe, et al., 1981, Ann. Surg. 194:472), and inhibition has been shown to suppress the development of tumors both in the ovary and in the testis (Matzuk, et al., 1992, Nature, 360:313).
Many of the members of the TGF-xcex2 family are also important mediators of tissue repair. TGF-xcex2 has been shown to have marked effects on the formation of collagen and causes of striking angiogenic response in the newborn mouse (Roberts, et al., 1986, Proc. Natl. Acad. Sci., USA 83:4167). The bone morphogenic proteins (BMPs) can induce new bone growth and are effective for the treatment of fractures and other skeletal defects (Glowacki, et al., 1981 Lancet, 1:959; Ferguson, et al., 1988, Clin. Orthoped. Relat. Res., 227:265; Johnson, et al., 1988, Clin. Orthoped. Relat. Res., 230:257).
C. Connective Tissue Growth Factor
A previously unknown growth factor, related to PDGF, and termed Connective Tissue Growth Factor (CTGF), has been reported in a related patent. See, U.S. Pat. No. 5,408,040. CTGF is a cysteine-rich mitogenic peptide which is selectively induced in fibroblasts after activation with TGF-xcex2. Igarashi, et al., 1993, Mol. Biol. Cell 4:637-645.
CTGF is a member of a family of peptides that include serum induced gene products ceflo (Simmons, et al., 1989, Proc. Natl. Acad. Sci. USA 86:1178-1182), cyr61 (O""Brien, et al., 1990, Mol. Cell. Biol. 10:3569-3577), fisp12/IG M1 (Ryseck, et al., 1993, Cell Growth and Differ. 2:225-233), and a chicken-transforming gene, nov (Joliot, et al. 1992, Mol. Cell Biol. 12:10-21 (1992). CTGF also shares sequence homology with a drosophila gene product, twisted gastrulation (twg) (Mason, et al.., 1994, Genes and Develop. 8:1489-1501), which determines cell fates during dorsal/ventral pattern formation in the embryo.
As reported in that patent, CTGF is the product of a distinct gene. As also reported in U.S. Pat. No. 5,408,040, CTGF possesses mitogenic activity. The ultimate result of this mitogenic activity in vivo, is the growth of targeted tissue. CTGF also possesses chemotactic activity, which is the chemically induced movement of cells as a result of interaction with particular molecules.
Although the molecule is antigenically related to PDGF, there is little if any peptide sequence homology between CTGF and PDGF. Anti-PDGF antibody has high affinity to the non-reduced forms of the PDGF isomers and the CTGF molecule and ten-fold less affinity to the reduced forms of these peptides, which lack biological activity.
A second protein, identified as xe2x80x9cconnective tissue growth factor-2xe2x80x9d or xe2x80x9cCTGF-2,xe2x80x9d has been reported also. See, PCT Application No. PCT/US94/07736 (International Publication No. WO 96/01896). According to the PCT Application, CTGF-2 may also be used to enhance the repair of connective and support tissue. Although identified as a connective tissue growth factor, CTGF-2 is not closely related to the CTGF of the present invention. Specifically, the CTGF family is comprised of three distinct groups of proteins: CTGF/Fisp12, cyr61 and nov. The protein of the claimed invention falls within the first group of proteins, as compared to CTGF-2, which falls with thecyr61 group. PCT Application No. PCT/US94/07736 at 4.
Notwithstanding the identification of various PDGF related growth factors, including CTGF, prior to the present invention, such factors have not been proven to be an effective induction agent for the production of matrices, including the induction of bone and/or cartilage tissue.
The subject invention provides novel methods and compositions for the treatment of diseases, disorders or ailments wherein matrix and/or connective tissue production, including the production of bone and/or cartilage, is desired. The subject invention is likewise directed to the treatment of diseases, disorders or ailments wherein the promotion of wound healing is desired.
More specifically, the compositions of the present invention comprise CTGF and/or fragments and/or derivatives thereof (hereinafter collectively xe2x80x9cCTGFxe2x80x9d), alone or in combination with other growth factors. The CTGF used in the subject compositions may be either obtained by isolation from natural sources, synthetic manufacture, or production by recombinant genetic engineering techniques.
In one aspect of the invention, the methods of the present invention comprise the administration of an effective amount of CTGF, alone or in combination with one or more compounds, to treat diseases, disorders or ailments wherein the induction of bone or cartilage tissue is desired. In a preferred embodiment of this method, such additional compound is a growth factor.
In another aspect of the invention, the methods of the present invention comprise the administration of an effective amount of CTGF, alone or in combination with one or more compounds, again preferably one or more growth factors, to treat diseases, disorders or ailments wherein the promotion of wound healing is desired.
In a preferred embodiment of the invention, the composition comprising CTGF is administered directly onto or into the site in which bone or cartilage induction is desired so as to induce the formation of such bone or cartilage. In another embodiment, the composition is formulated for targeted delivery or alternatively, are designed for the release of the novel compositions in the relevant site (e.g., the wound-in which cartilage formation is desired). In each case, the CTGF containing composition is appropriately formulated for administration to a patient in need.
As used in this Specification, the term xe2x80x9cCTGFxe2x80x9d shall mean: (1) a protein encoded by the amino acid sequence set forth at FIG. 1C, (2) a protein having CTGF activity wherein such protein is encoded by the amino acid sequence of FIG. 1C wherein one or more amino acids have been added, deleted, mutated, substituted or otherwise altered (xe2x80x9cderivativexe2x80x9d) and the nucleotide sequence encoding said protein can hybridize to the nucleic acid sequence of FIG. 1C under stringent conditions, or (3) a fragment of CTGF or a derivative thereof.
As used in this Specification, the term xe2x80x9cinduce,xe2x80x9d as used herein, shall mean to produce, form, to cause to produce, or to cause to form.
As used in this Specification, the phrase xe2x80x9cinduction agentxe2x80x9d shall mean an agent, including proteins or other biological materials, which causes the production or formation of a specific end result (e.g., the production of connective tissue).
As used in this Specification, the term xe2x80x9cpolynucleotidexe2x80x9d denotes DNA, cDNA and/or RNA which encode untranslated sequences which flank the structural gene encoding CTGF. For example, a polynucleotide of the invention includes 5xe2x80x2 regulatory nucleotide sequences and 3xe2x80x2 untranslated sequences associated with the CTGF structural gene. A polynucleotide of the invention which includes the 5xe2x80x2 and 3xe2x80x2 untranslated region is illustrated in FIG. 1C. The 5xe2x80x2 regulatory region, including the promoter, is illustrated in FIG. 1B. A more detailed description of the polynucleotides contemplated by the present invention may be found at U.S. Pat. No. 5,408,040.
As used in this Specification, the phrase xe2x80x9cstringent conditionsxe2x80x9d as used herein, refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50xc2x0 C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42xc2x0 C.; or (3) employ 50% formamide, 5xc3x97SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5xc3x97Denhardt""s solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42xc2x0 C., with washes at 42xc2x0 C. in 0.2xc3x97SSC and 0.1% SDS.
As used in this Specification, the phrase xe2x80x9crecombinant expression vectorxe2x80x9d refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the CTGF genetic sequences.
As used in this Specification, the phrase xe2x80x9ctherapeutically effectivexe2x80x9d means that amount of CTGF which is effective in inducing bone or cartilage formation or wound healing.